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1 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
2 Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, Canada
3 Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919, USA
Correspondence
Jens Walter
jwalter2{at}unl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Bacteria of the genus Lactobacillus are commonly detected as inhabitants of gut ecosystems and predominate in the proximal (gastric) regions of animals such as pigs, chickens, mice and rats (Tannock, 1992; Walter, 2005). The high population levels of lactobacilli in the gut of these animals are facilitated through bacterial adherence to the non-secretory, stratified, squamous epithelia present in the forestomach, pars oesophagea and crop of mice and rats, pigs and chickens, respectively. The epithelial associations formed by lactobacilli show characteristics of bacterial biofilms (Donlan & Costerton, 2002) because the bacteria are firmly attached to a surface (epithelium) and are embedded in a matrix of extracellular polymeric substances (Fuller & Brooker, 1974; Savage et al., 1968). The mechanisms by which lactobacilli form these epithelial associations are not well understood, but preliminary in vitro investigations have shown that carbohydrate molecules are likely to be involved, while a large surface protein (Lsp) appears to initiate adherence in vivo (Tannock, 1997; Walter et al., 2005).
Extracellular polysaccharides (exopolysaccharides, EPS) are synthesized by a wide variety of bacteria including lactic acid bacteria, and they have been shown to contribute to dental biofilm formation and cell aggregation of streptococci (Burne et al., 1996; Munro et al., 1991; Yamashita et al., 1993). Many strains of lactobacilli produce homopolysaccharides (HoPS) and oligosaccharides (OS) consisting of either glucose residues (glucans and gluco-oligosaccharides, GOS) or fructose residues (fructans and fructo-oligosaccharides, FOS) (Gänzle & Schwab, 2005; Korakli & Vogel, 2006; van Hijum et al., 2006). These compounds are synthesized from sucrose by the single action of extracellular enzymes termed glycosyltransferases, or more specifically glucosyltransferases and fructosyltransferases, respectively. Research on EPS of lactobacilli has focused on their properties as viscosifying, stabilizing, emulsifying, gelling, water-binding and prebiotic agents in the food industry (Bello et al., 2001; Korakli & Vogel, 2006; van Hijum et al., 2006). However, whereas the importance of streptococcal glycosyltransferases for colonization of the oral cavity is clearly established, the ecological importance of these enzymes and their products has not been revealed for lactobacilli (Korakli & Vogel, 2006).
Strains of Lactobacillus reuteri are commonly detected in the gastrointestinal tract of humans, pigs, chickens, mice and rats (Reuter, 2001; Walter, 2005). They often produce glucans and fructans of different linkage types, and some glycosyltransferases responsible for their production have been biochemically characterized (Kralj et al., 2002, 2004; Tieking et al., 2005; van Hijum et al., 2002, 2006). HoPS and OS formation of L. reuteri TMW1.106 and LTH5448 and its regulation under different environmental conditions has been previously investigated in detail (Gänzle & Schwab, 2005; Schwab & Gänzle, 2006; Schwab et al., 2007). L. reuteri TMW1.106 forms large amounts of a high molecular mass glucan and GOS and low amounts of FOS from sucrose and expresses the gtfA and inu genes encoding a glucosyltransferase and an inulosucrase, respectively (Schwab & Gänzle, 2006; Tieking et al., 2003). L. reuteri LTH5448 produces a high molecular mass fructan and FOS and expresses the ftfA gene encoding a levansucrase (Schwab & Gänzle, 2006). Insertional inactivation of these genes eliminated the synthesis of corresponding poly- and oligosaccharides in L. reuteri TMW1.106 and L. reuteri LTH5448, but did not impair the growth and metabolism of maltose and glucose (Schwab et al., 2007).
The prevalence of HoPS and OS production among lactobacilli isolated from gut ecosystems and the importance of these compounds in cell aggregation and biofilm formation of bacteria indigenous to the oral cavity led us to hypothesize that EPS production might constitute an important phenotypic trait of lactobacilli in colonization of the gastrointestinal tract. The availability of well-characterized, isogenic gtfA, inu and ftfA mutants of L. reuteri (Schwab et al., 2007) and the Lactobacillus-free mouse model (Tannock et al., 1988) provided an excellent opportunity to test this hypothesis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The mutants were derived by insertional mutagenesis of genes encoding the respective glycosyltransferases by site-specific integration of pORI28 into the chromosome (Schwab et al., 2007). Cultures were propagated anaerobically at 37 °C in Lactobacilli MRS medium (Difco) unless otherwise stated. Erythromycin (5 µg ml–1) was added to the growth medium for culture and maintenance of the mutant strains. Given that the plasmid insertion of the mutants showed high stability in the absence of antibiotics (Schwab et al., 2007), functional assays and phenotypic comparisons were performed using media without erythromycin. Bacterial growth in vitro was investigated by monitoring optical densities (OD600) as described previously (Tannock et al., 2005).
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were grown for 16 h (0.5 % inoculum) in either MRS medium or MRS medium supplemented with 1 % (w/v) sucrose. Cells were harvested from 1 ml of culture by centrifugation at 6000 g for 7 min at room temperature, washed once in 1 ml phosphate-buffered saline (PBS) adjusted to either pH 4 or 7, and resuspended in the initial volume of the same solution. Cell suspensions were kept at room temperature and autoaggregation was monitored visually. For determination of coaggregation, two tubes containing 1 ml cell suspension were prepared as described above using PBS (pH 4). Then 100 µl of a 10x concentrated cell suspension of the second strain was added to one of the tubes. After 1 h at room temperature, the optical densities were determined in both tubes. The degree of coaggregation between the two strains was recorded as the difference in optical densities following addition of cells of the second strain.
Biofilm experiments.
L. reuteri TMW1.106, the gtfA mutant and the inu mutant were grown for 12 h in MRS medium. Cells were harvested from 0.5 ml culture by centrifugation at 6000 g for 7 min at room temperature and resuspended in 1 ml prewarmed (37 °C) half-strength MRS medium containing 0.5 % sucrose (pH 4.5). Disposable flow cells (Stovall Life Science) were connected to a sterile reservoir of the same medium stored at 37 °C. Flow was generated using a Barnant Manostat Multichannel Pump (Carter). The flow rate was adjusted individually for each channel to 12 ml h–1. The flow condition used was at least 50 times faster than the doubling time of L. reuteri TMW1.106. Medium flow was stopped, and the flow cell was aseptically inoculated with bacterial cultures using sterile syringes as described by the manufacturer of the flow cell. After 1 h, flow was started again, and biofilm formation was monitored at different time points using a Nikon Optiphot microscope. Images of mature biofilms were acquired using an Olympus SZX12 dissecting microscope.
Experiments with mice.
Lactobacillus-free mice (males and females) were maintained throughout the experiments in isolators using gnotobiotic technology (Tannock et al., 1988). The Lactobacillus-free status was verified on a regular basis. The animals had free access to water and a standard rat diet (NRM Diet 86, Tegel). The feed contained about 1.25 % sucrose from molasses. The animals were 3–6 weeks of age at the start of the experiments. All animal experiments were conducted with approval from the University of Otago Animal Ethics Committee (approval number 104/02).
Testing the ability of L. reuteri TMW1.106 and LTH5448 to colonize Lactobacillus-free mice.
The ability of L. reuteri strains TMW1.106 and LTH5448 to colonize the gastrointestinal tract of Lactobacillus-free mice was tested by distributing 10 ml of overnight culture (
5x109 bacteria) per cage over the feed and fur of the animals (three mice per bacterial strain). Animals were killed 14 days later, and gut samples (forestomach, jejunum, caecum) were collected for bacteriological culture of serial dilutions of organ homogenates on Rogosa SL agar (Difco), as described previously (Walter et al., 2003).
Ecological competitiveness of the gtfA, inu and ftfA mutants.
Measurements of ecological competitiveness of strains were made by inoculating Lactobacillus-free mice (intragastric gavage) with 1 : 1 mixtures (total dose of 106 lactobacilli) of mutant and respective wild-type strains as described previously (Walter et al., 2005). Quantification of the mutant strain and the total Lactobacillus populations in faecal (after 7 days colonization), forestomach and caecal (14 days colonization) samples was achieved by determining counts on Rogosa SL agar plates with or without erythromycin as described previously (Walter et al., 2005). In competition experiments with L. reuteri TMW1.106 and Lactobacillus johnsonii #21 (both erythromycin-sensitive), differentiation of strains was achieved by plating gut samples (forestomach and caecum) after 7 days on Rogosa SL agar containing 0.05 % bromocresol green (Sigma). Each strain was quantified based on its distinctive colony morphologies. Correct differentiation of TMW1.106 and #21 was confirmed by picking representative colonies to MRS agar supplemented with 5 % sucrose, where TMW1.106 but not #21 produced large amounts of EPS (slimy phenotype). Ninety-eight per cent of colonies were identified correctly based on colony morphology on bromocresol green agar.
Experiments in Lactobacillus-free mice with pure cultures of L. reuteri TMW1.106, gtfA mutant and inu mutant to study colonization dynamics, in vivo metabolism and biofilm formation.
Lactobacillus-free mice were inoculated by intragastric gavage with pure cultures (dose of 105 lactobacilli) of either TMW1.106, or the gtfA or inu mutant strain. Bacterial populations were quantified in faecal samples 2 days after inoculation by plating dilutions of homogenates on Rogosa SL agar (Difco) with or without erythromycin. The animals were killed 7 days after inoculation, and the size of Lactobacillus populations in the caecum was determined. Comparison of colony counts on media with or without erythromycin indicated that the mutational insert of both mutants was stably maintained throughout the experiment (data not shown).
Stomach contents of seven Lactobacillus-free mice and of five mice colonized for 7 days by either L. reuteri TMW1.106 or the gtfA or inu mutant were collected, pooled and freeze-dried. Carbohydrates in stomach contents were extracted using double-distilled H2O at 80 °C for 2 h. Maltose, glucose and lactate were separated using an Aminex HPX 87H column (Bio-Rad) with 5 mM H2SO4 as solvent at 0.4 ml min–1 and detected with a refractive index detector as described previously (Schwab et al., 2007).
Bacterial associations formed on the epithelial surface of the murine forestomach were investigated by transmission electron microscopy (TEM) as described previously (Walter et al., 2007). Briefly, stomachs from two mice colonized by either L. reuteri TMW1.106, the inu mutant or the gtfA mutant (see above), excised from the animals, were fixed in a 0.1 M cacodylate buffer (pH 7) containing 3 mg ruthenium red ml–1 and 3 % glutaraldehyde for 4 h at room temperature and then at 4 °C for 4–5 days. Forestomach pieces of 1 mm3 were successively fixed, dried and washed before embedding in resin. Sections of 80 nm thickness were examined by TEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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whose cells autoaggregated. At pH 7, cells aggregated whether sucrose was present in the growth medium or not, and autoaggregation was not affected by inactivation of the gtfA and inu genes (data not shown). At pH 4, L. reuteri TMW1.106 cells aggregated when grown with sucrose, and autoaggregation of the gtfA and the inu mutant was adversely affected. Aggregation of the inu mutant cells was slower, while the gtfA mutant did not aggregate (Fig. 1a, b). None of the strains aggregated at pH 4 when grown in the absence of sucrose, but autoaggregation could be initiated for L. reuteri TMW1.106 and the inu mutant, but not for the gtfA mutant, by the addition of 
1 mg sucrose ml–1 to the cell suspensions and incubation at 37 °C for 5 h (data not shown). These findings indicated that glucan production mediated by GtfA was involved in autoaggregation of TMW1.106 cells.
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). Therefore, gtfA cells could still coaggregate with L. reuteri TMW1.106 and the inu mutant. The ability of the gtfA mutant to coaggregate was not dependent on sucrose, but required sucrose in the growth medium of the wild-type strain (data not shown).
Biofilms in the proximal gut are composed of different Lactobacillus strains and species. Therefore the effect of mutation of gtfA and inu on coaggregation with other strains of L. reuteri (LTH5448, its ftfA mutant and 100-23) and L. johnsonii (strains #21 and 100-100) was tested. All the strains coaggregated with L. reuteri TMW1.106 at pH 4, resulting in a reduction in optical density (Fig. 1c). Inactivation of gtfA, but not inu, adversely affected coaggregation with strains of L. reuteri. Inactivation of ftfA of L. reuteri LTH5448 had no effect. Coaggregation of L. reuteri TMW1.106 with strains of L. johnsonii (#21 and 100-100) was not affected by inactivation of gtfA.
Evaluation of in vitro biofilm formation of L. reuteri TMW1.106, the gtfA mutant and the inu mutant
Biofilm formation was assessed by direct, non-destructive examination of the cells adhering to a glass surface. As shown in Fig. 2(a), wild-type TMW1.106 formed a biofilm that was visible under a light microscope within 18 h. The gtfA and the inu mutant showed impaired biofilm formation. Intense cell aggregation could be observed for the wild-type and the inu mutant. In contrast, cell aggregation of the gtfA mutant was virtually absent at 12 h, and greatly reduced at 18 h. Biofilms became macroscopically visible after around 30 h. When observed with a dissecting microscope, the biofilms formed by the mutants showed reduced density compared to the wild-type (Fig. 2b). Although clearly impaired when compared with the wild-type, the inu mutant performed slightly better than the gtfA mutant in two independent experiments (Fig. 2).
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; Walter et al., 2003, 2005, 2007). Further animal experiments were carried out to determine whether the L. reuteri glycosyltransferase mutant strains exhibit an impaired competitiveness in the murine gut. Competitive colonization experiments using 1 : 1 mixtures of wild-type and mutant strains as inoculum (n=9–11) revealed that the inu mutant competed poorly with L. reuteri TMW1.106 (see Fig. 3b). The gtfA and ftfA mutant populations were not different from that of the wild-type TMW1.106 and LTH5448, respectively (Fig. 3a, c).
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Metabolism of lactobacilli during growth in the stomach of mice
Lactobacilli inhabiting the forestomach utilized maltose and glucose and produced lactate (Table 2). Sucrose was present in the stomach of mice not colonized with lactobacilli but the peak was too small for quantification (data not shown). Interestingly, stomach contents of mice colonized with the inu mutant contained higher maltose and lower lactate concentrations compared to stomach contents of mice colonized with the wild-type or the gtfA mutant, indicating that the utilization of maltose by the inu mutant was reduced in vivo. We compared the in vitro growth of L. reuteri TMW1.106, the gtfA and the inu mutant in MRS broth containing glucose or maltose as the only carbohydrates, but the mutant strains did not differ from the wild-type with respect to growth rate (data not shown).
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). The organisms formed cell layers and aggregates on the epithelium, and bacterial densities were highest in inversions (infoldings) of the epithelium and on epithelial cells that had been shed into the lumen. The gtfA mutant and the inu mutant formed epithelial associations that were qualitatively similar to the wild-type (Fig. 4b, c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Here we present evidence that gtfA and inu contribute to cell aggregation and in vitro biofilm formation of L. reuteri TMW1.106. Furthermore, by using the well-characterized Lactobacillus-free mouse model, we could show that both genes contribute to the ecological performance of the organism during gut colonization. In contrast, inactivation of ftfA did not affect coaggregation or ecological performance of L. reuteri LTH5448 in the experiments presented here.
According to Rickard et al. (2003), bacterial aggregation is an integral process of biofilm formation which proceeds in the form of a succession of adhesion and multiplication events. Autoaggregation of L. reuteri TMW1.106 at neutral pH was independent of sucrose, and inactivation of gtfA and inu had no effect. In contrast, under acidic conditions (pH 4), autoaggregation required both sucrose and GtfA. These findings indicate that the glucan produced by GtfA from sucrose mediates aggregation of L. reuteri TMW1.106. Glucan production has been shown to play an important role in cell aggregation in Gram-positive bacteria (Banas & Vickerman, 2003; Gibbons & Fitzgerald, 1969; Lynch et al., 2007). A possible explanation for the impaired aggregation of the inu mutant is that Inu functions as a glucan-binding protein in L. reuteri TMW1.106. Accordingly, homologues of Inu function as glucan-binding proteins in Streptococcus mutans (Rozen et al., 2004; Russell et al., 1983). The gtfA mutant could still coaggregate with wild-type cells that had been grown in medium containing sucrose, showing that the ability to bind glucan was not mediated by GtfA. Coaggregation with non-aggregating strains of L. reuteri (100-23, LTH5448) was adversely affected (but not eliminated) by inactivation of gtfA. These findings indicated that some L. reuteri strains possess glucan-binding proteins that contribute to coaggregation, even though they do not produce glucan.
We tested the ability of L. reuteri TMW1.106, the gtfA mutant and the inu mutant to form biofilms under acidic conditions (pH 4.5) on a glass surface. Biofilm formation was adversely affected for both mutants. Microscopic evaluation revealed that cell aggregation on the glass slides was basically absent for the gtfA mutant after 12 h, while the inu mutant showed clear signs of cell aggregation. These findings are consistent with those obtained in the aggregation tests. We conclude that GtfA and Inu contribute to biofilm formation by allowing individual cells to form cell aggregates.
Due to production of lactic acid by the bacteria and host acid secretion, the milieu encountered by lactobacilli during colonization of the forestomach is likely to be acidic (Baumgartner & Montrose, 2004). The lactate levels in the forestomach of mice (Table 2) correspond to those that are typical for stationary cultures of lactobacilli on cereal substrates, which show a pH of around 4 (Gänzle et al., 1998). The importance of GtfA and Inu for cell aggregation and biofilm formation under acidic conditions therefore provides a potential explanation for the decreased ecological performance of the two mutant strains when colonizing the gut of Lactobacillus-free mice. As shown in Fig. 4, L. reuteri TMW1.106 forms epithelial associations that are characterized by the formation of cell aggregates while colonizing the forestomach. These formations resemble natural biofilms detected on forestomach (rodents), crop (chicken), and pars oesophagea (pigs) epithelia, which are dominated by lactobacilli (Fuller & Brooker, 1974; Fuller et al., 1978; Savage et al., 1968). Although the gtfA and inu mutants were still able to form epithelial associations that were qualitatively similar to the wild-type, our in vitro data imply that GtfA and Inu enhance the ability to form these cell aggregates on the forestomach epithelium.
A striking finding of this study was that the disruption of inu impaired colonization of mice by L. reuteri TMW1.106 in competition experiments using the wild-type strain, while disruption of gtfA had no adverse effect. On the other hand, GtfA did contribute to ecological performance when TMW1.106 colonized the gut alone or in competition with L. johnsonii #21. Thus both enzymes contribute to the colonization phenotype of L. reuteri TMW1.106 but they appear to play different roles. A possible explanation is that Inu is a glucan-binding protein and a receptor for the glucan produced by GtfA. Inactivation of Inu would then impair binding to the glucan matrix of the biofilm, resulting in decreased ecological performance in competition experiments with the wild-type. In contrast, assuming that GtfA is only involved in glucan synthesis but not in binding, the gtfA mutant would remain competitive when extracellular glucan is provided by the wild-type strain. Determination of the exact role of GtfA and Inu in cell aggregation and biofilm formation requires further experimentation.
Although we clearly show the importance of GtfA and Inu for cell aggregation and biofilm formation, we cannot exclude other functions for these enzymes in the murine gut. Previous work showed that glycosyltransferases of L. reuteri could have several functions under different environmental conditions (Gänzle & Schwab, 2005; Schwab et al., 2007). For example, the gtfA and inu mutants showed a lower resistance to lactic acid when compared to wild-type TMW1.106 (Schwab et al., 2007). This could account for the reduced metabolic turnover of the inu mutant in the murine stomach (Table 2). However, growth rates of both mutants were not impaired in media containing maltose and glucose (Schwab et al., 2007), which are the main growth substrates available in the gut, and the preferred carbon sources for L. reuteri.
In conclusion, our study has shown the importance of two EPS-producing enzymes, GtfA and Inu, for the ecological performance of a gut commensal. Moreover, this is to our knowledge the first report on the importance of glycosyltransferases for cell aggregation and biofilm formation in the genus Lactobacillus. A high proportion of Lactobacillus isolates from gut environments produce EPS and possess orthologues of GtfA and Inu (Gänzle & Schwab, 2005; Korakli & Vogel, 2006; Tieking et al., 2003, 2005; van Hijum et al., 2006). Taken together, this study and the literature data indicate that GtfA and Inu confer important ecological attributes on L. reuteri TMW1.106 and contribute to colonization of the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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Edited by: P. W. O'Toole
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Received 14 June 2007;
revised 14 September 2007;
accepted 16 October 2007.
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